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目的:运用分子生物学技术克隆CYP 4A1全长cDNA基因,进一步构建可由强力霉素诱导表达CYP4A1的逆转录病毒重组质粒。方法:运用逆转录-聚合酶链反应(RT-PCR)从大鼠肝组织中扩增出CYP 4A1全长基因,克隆至pMD18-T载体,将构建正确的pMD18-CYP 4A1质粒采用双酶切亚克隆到逆转录病毒载体载体pRe-vTRE中,采用酶切反应、PCR鉴定和序列测定鉴定重组逆转录病毒载体pRevTRE/CYP 4A1;在脂质体介导下分别将质粒pRevTRE/CYP 4A1与逆转录病毒四环素调节质粒pRevTet-on转染至包装细胞PT67,分别经抗性筛选获得稳定产生病毒的包装细胞系后收集转染后的细胞上清。用荧光定量PCR测定病毒滴度。结果:经酶切鉴定、PCR鉴定和序列测定证实成功构建了pRevTRE/CYP 4A1逆转录病毒重组质粒;转染pRevTet-on的PT67细胞病毒滴度为7.2×1010copies/L,转染pRevTRE/CYP 4A1的PT67细胞病毒滴度为5.46×109copies/L。结论:成功构建了含四环素调控CYP 4A1基因的逆转录病毒重组质粒,建立了能产较高滴度逆转录病毒的包装细胞系,为CYP 4A1基因功能及其相关疾病的研究打下实验基础。
OBJECTIVE: To clone full-length cDNA of CYP 4A1 using molecular biology techniques and construct recombinant retroviral vector expressing CYP4A1 induced by doxycycline. Methods: The full length CYP4A1 gene was amplified from rat liver tissue by reverse transcription polymerase chain reaction (RT-PCR) and cloned into pMD18-T vector. The constructed plasmid pMD18-CYP 4A1 was digested by double enzyme digestion The recombinant retroviral vector pRevTRE / CYP 4A1 was identified by restriction enzyme digestion, PCR and sequence analysis. The recombinant plasmid pRevTRE / CYP 4A1 was reverse transcribed The tetracycline-regulated plasmid pRevTet-on was transfected into the packaging cell PT67, and the stable virus-producing packaging cell lines were obtained after resistance screening. The transfected cell supernatants were collected. The virus titer was determined by fluorescence quantitative PCR. Results: The recombinant plasmid pRevTRE / CYP 4A1 was confirmed by restriction enzyme digestion, PCR and sequencing. The titer of PT67 virus transfected with pRevTet-on was 7.2 × 1010 copies / L and transfected with pRevTRE / CYP 4A1 The PT67 cell virus titer was 5.46 × 109 copies / L. CONCLUSION: The retroviral recombinant plasmid containing tetracycline-regulated CYP 4A1 gene was successfully constructed and a packaging cell line capable of producing high titer retroviruses was established, laying an experimental foundation for the study of CYP 4A1 gene function and its related diseases.